Mixtures derived from grains of eugenia jambolana lamarck, preparation and use of said mixtures and some of their constituents as medicaments

ABSTRACT

PCT No. PCT/FR97/00207 Sec. 371 Date Aug. 6, 1998 Sec. 102(e) Date Aug. 6, 1996 PCT Filed Feb. 3, 1997 PCT Pub. No. WO97/28813 PCT Pub. Date Aug. 14, 1997The present invention relates to mixtures which can be isolated from grains of Eugenis Jambolana Lamarck, the preparation of such mixtures, the medicaments containing said mixtures or constituents of said mixtures, and the use of these mixtures and constituents for the preparation of a medicament.

The present invention relates to mixtures which can be isolated fromEugenia Jambolana Lamarck seeds (Myrtaceae family), medicamentscontaining these mixtures or some of their constituents, the use ofthese mixtures and constituents for the preparation of an antidiabeticmedicament and their preparations.

A plant extract prepared from Eugenia Jambolana seeds or bark containinga polyphenol and sterol mixed complex is described in patent FR2,465,484.

New mixtures which can be isolated from Eugenia Jambolana Lamarck seeds,and which are free of polyphenol and sterol complex, as well as certainconstituents of these mixtures endowed with hypoglycaemic properties,have now been found.

These mixtures are characterized in that they are free of polyphenol andsterol derivatives and can be isolated by grinding Eugenia JambolanaLamarck seeds, maceration of the powder with a lower aliphatic alcoholwith the use of heat, filtration, recovery of the insoluble part nolonger containing polyphenol and sterol compounds, treatment of theinsoluble part with an ammoniacal solution, treatment of the ammoniacalmixture with a lower aliphatic alcohol with the use of heat, filtration,recovery of the insoluble matter and drying this insoluble matter whichconstitutes the mixture I, then optionally treatment of the mixture Iwith a water-lower aliphatic alcohol solution, filtration, partialconcentration of the filtrate, purification on nonpolar adsorbentresins, partial concentration, centrifugation, ultrafiltration andisolation of the mixture II.

From the mixture II, there may also be separated sodium oxamate and thecompounds of formula: ##STR1## in which either R₁ represents a hydrogenatom and R₂ represents a chain of formula: ##STR2## or R₁ represents achain of formula (A) and R₂ represents a hydrogen atom.

Sodium oxamate has already been described by TOUSSAINT, Ann., 120, 237(1861).

The compounds of formula (I) have already been described by KUHN et al.,Ann., 644, 122-127 (1961); TSUCHIDA et al., Agr. Biol. Chem., 39 (5),1143-1148 (1975); TSUCHIDA et al., Agr. Biol. Chem., 40 (5), 921-925(1976); TSUCHIDA et al., Nippon Shokuhin Kogyo Gakkaishi, 37, 154-161(1990) and AVALOS et al., tetrahedron, 49, 2655-2675 (1993).

The present invention also relates to the process for preparing themixture I from dried and finely ground Eugenia Jambolana Lamarck seeds.

The powder is screened, preferably with the aid of a normalized screenwith holes 0.5 μm in diameter and then subjected to the followingtreatment:

a--maceration, with stirring, in a lower aliphatic alcohol, at atemperature of between 40 and 70° C.,

b--filtration under vacuum and recovery of the insoluble matter,

c--maceration, with stirring, of the insoluble matter with a loweraliphatic alcohol at a temperature of between 40 and 70° C.,

d--filtration under vacuum and removal of the alcoholic phasescontaining mainly the undesirable polyphenols and sterols,

e--taking up the insoluble matter in an ammoniacal solution at atemperature of between 10 and 30° C.,

f--taking up the whole wet ammoniacal mass in a water-lower aliphaticalcohol solution, at a temperature of between 40 and 70° C.,

g--filtration and removal of the alcoholic solution,

h--washing of the insoluble matter with a lower aliphatic alcohol,filtration and removal of the alcoholic solution,

i--recovery of the insoluble matter and drying.

In step a, the procedure is generally carried out by means of 2 to 10liters of a lower aliphatic alcohol such as methanol or ethanol per 1 kgof screened powder. Preferably, 5 liters of ethanol with a titre of93-95° Gay Lussac are used at 60° C. for 1 hour.

The filtration of step b is preferably carried out under a vacuum of 40kPa.

In step c, the procedure is generally carried out by means of 2 to 10liters of a lower aliphatic alcohol such as methanol or ethanol per 1 kgof starting screened powder. Preferably, 4 liters of ethanol with atitre of 93-95° Gay Lussac are used at a temperature of 60° C. for 1hour.

The filtration of step d is preferably carried out under a vacuum of 40kPa.

In step e, per 1 kg of starting screened powder, 750 to 1250 ml of anaqueous ammoniacal solution preferably containing 350 ml of 28% ammoniumhydroxide per 1000 ml are generally used. It is particularlyadvantageous to use 1 liter of the aqueous ammoniacal solution and tocarry out the procedure for 10 to 30 hours and, preferably, 20 hours ata temperature close to 20° C.

In step f, the wet ammoniacal mass obtained from 1 kg of startingscreened powder is generally taken up, with stirring, in 2 to 10 litersof a lower aliphatic alcohol-water mixture (methanol or ethanol forexample) (70/30 to 80/20 by volume) and, preferably, in 5 liters of anethanol-water mixture (75/25 by volume), at 60° C., for 1 hour.

In step g, the filtration is preferably carried out on a cotton clothand under a vacuum of about 80 kPa.

In step h, the washing is generally carried out with 500 to 1500 ml of alower aliphatic alcohol (methanol or ethanol for example) per 1 kg ofstarting screened powder and, preferably, with 1 liter of ethanol andthe filtration is carried out on a cotton cloth and under a vacuum ofabout 80 kPa.

In step i, the drying is preferably carried out in the open air andprotected from light.

The present invention also relates to the process for preparing themixture II.

The mixture I obtained above is subjected to the following operations:

j--treatment of the mixture I by means of a water-lower aliphaticalcohol solution,

k--decantation and then, on the one hand, drawing off the top phasewhich is filtered to give the filtrate 1 and, on the other hand,treating the bottom phase with water and filtration to give the filtrate2, pooling of the filtrates 1 and 2 and concentration to aqueous phase,

l--treatment with a nonpolar adsorbent resin and then filtration,

m--concentration of the filtrate, filtration and then ultrafiltration,

n--freeze-drying and isolation of the extract II.

In step j, 10 to 25 liters of the water-lower aliphatic alcohol solution(methanol or ethanol for example) (95/5 to 90/10 by volume) aregenerally used per 1 kg of the mixture I. It is preferable to carry outthe procedure in 18 liters of a water-ethanol solution (17.7-0.93 byvolume).

In step k, it is preferable to filter the top phase on a cotton cloth.It is advantageous to add, per 1 kg of the mixture I, 10 to 25 liters ofwater to the bottom phase and in particular 10 liters and to filter onsintered glass.

In step k, the concentration is generally carried out in a thermosiphonconcentrator at a temperature of 35° C. under a vacuum of 0.4 kPa.

In step 1, S861 resin or XAD-type resins marketed by Rhom and Hass arepreferably used and the mixture is filtered on sintered glass.

In step m, the concentration is generally carried out in a thermosiphonconcentrator at a temperature of 35° C. under a vacuum of 0.4 kPa. It isalso advantageous to carry out 3 successive ultrafiltrations on 10 kd, 3kd and 1 kd cartridges.

The present invention also relates to the process for preparing sodiumoxamate and the compounds of formula (I).

The said process consists in subjecting the mixture II to the followingoperations:

o--chromatography of the mixture II on an infusorial earth column,recovery of the fractions containing the 4 products and pooling of thesefractions into a single fraction,

p--chromatography of the fraction previously obtained on a Sephadex®column in order to obtain sodium oxamate, the compound of formula (I)for which R₁ represents a hydrogen atom and R₂ represents a residue (B)and a mixture of the compound of formula (I) for which R₁ represents ahydrogen atom and R₂ represents a residue (A) and of the compound offormula (I) for which R₁ represents a residue (A) and R₂ represents ahydrogen atom,

q--optionally, chromatography of the mixture of the compound of formula(I) for which R₁ represents a hydrogen atom and R₂ represents a residue(A) and of the compound of formula (I) for which R₁ represents a residue(B) and R₂ represents a hydrogen atom by HPLC.

The chromatography of step o is carried out by means of an organicsolvent such as heptane, ethyl acetate or a lower aliphatic alcohol.Preferably, a CHEM ELUT® column marketed by Prolabo is used, saturatedwith water and then eluted successively with heptane, a heptane-ethylacetate mixture (50/50 by volume), ethyl acetate, an ethylacetate-n-butanol mixture (95/5; 90/10; 80/20; 50/50; 20/80), n-butanol,and an n-butanol-water mixture (98/2 and then 95/5 by volume).

The chromatography of step p is preferably carried out by means of awater-ethanol mixture (50/50 by volume).

The chromatography of step q is generally carried out on a YMC 180DS-AQcolumn marketed by AIT with, as eluent, a water containing 0.1% offormic acid mixture.

In the preceding definitions and those which follow, the lower aliphaticalcohols preferably contain 1 to 4 carbon atoms.

The medicaments containing the mixtures I or II or sodium oxamate or oneor more compounds of formula (I) generally form part of the invention.

The present invention also relates to the use of the mixtures I and II,of sodium oxamate and of the compounds of formula (I) or a mixture ofthese with the preparation of medicaments for the treatment orprevention of diabetes and of the complications of diabetes.

The following examples illustrate the invention.

EXAMPLE 1

Preparation of the Mixture I

Eugenia Jambolana Lamarck seeds are dried in the open air, protectedfrom light, and then finely ground. The powder thus obtained is screenedwith the aid of a screen of mesh 0.5 μm. 1 kg of screened powder ismacerated, with mechanical stirring, in 5 liters of ethanol with a titreof 93-95° Gay Lussac, for one hour at 60° C. After filtration undervacuum, the insoluble matter is further treated under the sameconditions with 4 liters of ethanol of the same titre at 60° C. for afurther one hour. The two ethanolic extracts containing mainly theundesirable polyphenol and sterol complex are removed. After completefiltration, the insoluble matter is taken up in 1 liter of ammoniacalsolution (28% NH₄ OH 350 ml and distilled H₂ O in sufficient quantity toobtain 1 liter of solution) and the mixture is left in contact for about20 hours at a temperature close to 20° C. The wet ammoniacal mass isallowed to macerate again in 5 liters of the ethanol of titre 93 to 95°Gay Lussac-water mixture (75/25 by volume), with mechanical stirring,for 1 hour at 60° C. The insoluble matter is filtered and it is washedwith 1 liter of ethanol of the same titre; the filtrate and the washingsare removed. The final insoluble matter is dried in the open air andprotected from light. The mixture I is thus obtained in the form of apowder which is free of polyphenol and sterol derivatives. The yieldfrom the pulverized and screened seeds is 80%.

EXAMPLE 2

Preparation of the Mixture II

1 kg of the mixture I obtained in Example 1, in a solution containing17.7 liters of water and 0.93 liter of ethanol, is stirred for 3 hours.After decantation overnight, on the one hand, the top phase (13.5liters) is drawn off and then filtered on a cotton cloth in a 7-literfilter (Schott) to give the filtrate 1 (13.5 liters) and, on the otherhand, the bottom phase is stirred for 1 hour with 20 liters of water,filtered on No. 3 sintered glass to give the filtrate 2 (24 liters). Thefiltrates 1 and 2 are pooled and then concentrated in aqueous phase(33.5 liters) in a thermosiphon concentrator (Schott) at 35° C. under avacuum of 0.4 kPa. The concentrate is stirred for one hour with 2.5liters of S861 resin (Rhom and Haas) and then filtered on No. 3 sinteredglass. The filtrate is concentrated in a thermosiphon concentrator(Schott) at 35° C. and then under a vacuum of 0.4 kPa to 5.5 liters. Theconcentrate is centrifuged in a tubular centrifuge (centrifugal force62000 g) and filtered on a 0.22 μm filter (Gelman suporcap 100 type)pump. After 3 successive ultrafiltrations on 10 Kd, 3 Kd and 1 Kdcartridges and freeze-drying, 25 g of the mixture II are obtained in theform of a dark brown hygroscopic powder.

EXAMPLE 3

Constituents Obtained from the Mixture II

525 mg of the mixture II obtained in Example 2 in solution in 1.1 ml ofmilli Q-filtered water are chromatographed on a CHEM ELUT® columnmarketed by PROLABO, 50 cm in height and with a diameter of 1 cm,saturated with milli Q-filtered water. The elution is carried out withheptane using increasing gradients of ethyl acetate and then ofn-butanol, of n-butanol/hydrochloric acid and of water (fraction 1:heptane; fraction 2: heptane/ethyl acetate (50/50 by volume); fractions3-4: ethyl acetate; fractions 5-6 ethyl acetate/n-butanol (95/5 byvolume), fractions 7-8: ethyl acetate/n-butanol (90/10 by volume),fractions 9-10: ethyl acetate/n-butanol (80/20 by volume); fractions11-12: ethyl acetate/n-butanol (50/50 by volume), fractions 13-14: ethylacetate/n-butanol (20/80 by volume), fractions 15-16: n-butanol,fractions 17-18: n-butanol/water (98/2 by volume), fractions 19-21:n-butanol/water (95/5 by volume). 50 ml fractions are collected.Fractions 19 to 21 are combined and concentrated to dryness underreduced pressure. 108 mg of a fraction is thus obtained which ischromatographed on a Sephadex® LH20 column marketed by Pharmacia (height100 cm, diameter 1 cm) produced with a methanol-water mixture (50-50 byvolume). The elution is carried out with the same eluent mixture; 1 mlfractions are collected.

1--Fractions 88 to 91 are combined and concentrated to dryness. 14.4 mgof a product are obtained, which product gives, upon crystallizationfrom 0.5 ml of ethanol, 2 mg of 2,5-di-(tetrahydroxybutyl)pyrazine inthe form of white crystals whose characteristics are the following:

optical rotation [α]²⁰ (Na 589)=-137°±2.0 (dimethyl sulphoxide; c=0.5),

infrared spectrum produced on a Nicolet 60SX-R apparatus in solution inKBr, main characteristic absorption bands: 3281 cm⁻¹ (υ bound OH groupsincluding H₂ O), 2972+2940+2901+2880 cm⁻¹ (υ CH of the CH₂ and CHOHgroups), 2733 cm⁻¹ (υ bound OH groups), 1635 cm⁻¹ (deformations of theOH groups, including H₂ O), 1491 cm⁻¹ (υ C═C and C═N of the pyrazinenucleus), 1449 cm⁻¹ (υ C═C and C═N of the pyrazine nucleus+deformationsof the OH groups), 1413 cm⁻¹ (deformations of the OH groups), 1343 cm⁻¹(υ C═C and C═N of the pyrazine nucleus),1309+1290+1251+1215+1181+1161+1123 cm⁻¹ (deformations of the CH groups),1092 cm⁻¹ (υ CO of the secondary alcohols), 1048+1035 cm⁻¹ (υ CO of theprimary alcohols+pyrazine nucleus), 947+899+854 cm⁻¹ (secondaryalcohols), 877 cm⁻¹ (υ CH of the pyrazine nucleus), 727 cm⁻¹ (pyrazinenucleus+deformations of the OH groups), 639 cm⁻¹ (pyrazine nucleus),607+531 cm⁻¹ broad (deformations of the OH groups), 451+411 cm⁻¹(pyrazine nucleus),

mass spectrum performed on a FINNIGAN TSQ46 apparatus, mass ionizationmode M/z=321 (MH)+,

¹ H NMR spectrum (600 MHz, DMSO, chemical shift in ppm): 3.40 and 3.61(2 mts, 2H each: CH₂ at position 4', 4"); 3.58 (2 mts, 2H each: CH atposition 2', 2", 3', 3"); 4.36 (broad t, 2H: OH at position 4', 4");4.40 (d, J=7.2 Hz, 2H: OH at position 2', 2"); 4.63 (d, J=4.8 Hz, 2H: OHat position 3', 3"); 4.95 (dd J=6.0 and 0.6 Hz, 2H: CH at position 1',1"); 5.30 (d, J=6.0 Hz, 2H: OH at position 1', 1"); 8.61 (s, 2H, CH atposition 3 and 6),

ultraviolet spectrum: λ max=275 nm (ε=8260); 206 nm (ε=10220) (c=19mg/ml; water), λ max=276 nm (ε=7960); 206 nm (ε=9920) (c=19 mg/ml; HCl0.1N), λ max=275 nm (ε=7690); (c=19 mg/ml; KOH 0.1N),

HPLC on Y.M.C. 180DS-AQ column of 150×4.6 mm (batch AIT/DE940377)marketed by AIT, isocratic elution H₂ O +0.1% formic acid with a flowrate of 1 ml/min, UV detection at 270 nm, retention time: 2 min 32 s.

2--Fractions 92 and 93 are combined and concentrated to dryness. 9 mg ofa fraction are obtained, which fraction gives, upon crystallization from0.5 ml of ethanol, 1.2 mg of sodium oxamate having the samecharacteristics as those described by TOUSSAINT, Ann., 120, 237 (1861).

3--Fractions 94 to 97 are combined and concentrated to dryness. 25.9 mgof a fraction are obtained, which fraction gives, upon crystallizationfrom 0.5 ml of ethanol, 6.2 mg of a mixture of2-(tetrahydroxybutyl)-5-(2',3',4'-trihydroxybutyl)pyrazine and2-(tetrahydroxybutyl)-6-(2',3',4'-trihydroxybutyl)pyrazine which isfurther separated by HPLC on a Y.M.C. 180DS-AQ column of 150×4.6 mm(batch AIT/DE940377); isocratic elution H₂ O +0.1% formic acid with aflow rate of 1 ml/min, UV detection at 270 nm.

5.2 mg of 2-(tetrahydroxybutyl)-5-(2,3',4'-trihydroxybutyl)pyrazine arethus obtained, which has the following characteristics:

optical rotation [α]²⁰ (Na 589)=-116.3°±1.7 (dimethyl sulphoxide;c=0.5),

infrared spectrum performed on a Nicolet 60SX-R apparatus in solution inKBr, main characteristic absorption bands at 3398 cm⁻¹ (υ bound OHgroups including H₂ O), 2951+2922+2891 cm⁻¹ (υ CH of the CHOH groups),2761 cm⁻¹ (υ bound OH groups), 1636 cm⁻¹ (deformations of the OH groups,including H₂ O), 1483+1463 cm⁻¹ (υ C═C and C═N of the pyrazine nucleus),1411 cm⁻¹ (deformation of the OH groups), 1367+1328+1270+1227+1191 cm⁻¹(deformation of the CH groups), 1071 cm⁻¹ (υ CO of the secondaryalcohols), 1041 cm⁻¹ (υ CO of the primary alcohols+pyrazine nucleus),943+897 cm⁻¹ (secondary alcohols), 869 cm⁻¹ (υ CH of the pyrazinenucleus), 645 cm⁻¹ (CH of the pyrazine nucleus), 607 cm⁻¹ broad(deformations of the OH groups), 446+409 cm⁻¹ (pyrazine nucleus),

mass spectrum performed on a FINNIGAN TSQ46 apparatus, mass ionizationmode M/z=305 (MH)+,

¹ H NMR spectrum (600 MHz, DMSO, chemical shift in ppm): 2.70 and 3.04(2 dd, J=9.0 and 15.0 Hz and J=3.0 and 15.0 Hz, 1H each: CH₂ at position1"); between 3.30 and 3.45 (mts, CH at position 3", 4', 4"); between3.53 and 3.65 (mts, 4H: CH at position 2', 3', 4', 4"), 3.73 (mt, 1H: CHat position 2"); 4.37 (broad t, 1H OH at position 4'); 4.42 (mt, 2H: OHat position 2' and 4"); 4.60 (d, J=6 Hz, 1H: OH at position 2"); 4.63(d, J=6 Hz, 1H: OH at 3'); 4.67 (d, J=6 Hz, 1H: OH at position 3"); 4.92(dd J=6.0 and 0.6 Hz, 1H: CH at position 1'); 5.30 (d, J=6.0 Hz, 1H: OHat position 1'); 8.39 (s, 1H: CH at position 6); 8.61 (s, 1H: CH atposition 3),

ultraviolet spectrum: λ max=276 nm (ε=7756); 206 nm (ε=8738) (c=19mg/ml; water), λ max=277 nm (ε=7218); 208 nm (ε=7171) (c=19 mg/ml; HCl0.1 N), λ max=276 nm (ε=7467) (c=19 mg/ml; KOH 0.1N),

HPLC on Y.M.C. 18ODS-AQ column of 150×4.6 mm (batch AIT/DE940377)marketed by AIT, isocratic elution H₂ O +0.1% formic acid with a flowrate of 1 ml/min, UV detection at 270 nm, retention time: 3 min 75 s and1 mg of 2-(tetrahydroxybutyl)-6-(2',3',4'-trihydroxybutyl)pyrazine whichhas the following characteristics:

¹ H NMR spectrum (600 MHz, DMSO, chemical shift in ppm): 2.70 and 3.04(2 dd, J=9.0 and 15.0 Hz and J=3.0 and 15.0 Hz, 1H each: CH₂ at position1"); between 3.30 and 3.45 (mts, CH at position 3", 4', 4"); between3.53 and 3.65 (mts, 4H: CH at position 2', 3', 4', 4"), 3.73 (mt, 1H: CHat position 2"); 4.37 (broad t, 1H OH at position 4'); 4.42 (mt, 2H: OHat position 2' and 4"); 4.60 (d, J=6 Hz, 1H: OH at position 2"); 4.63(d, J=6 Hz, 1H: OH at 3'); 4.67 (d, J=6 Hz, 1H: OH at position 3"); 4.92(dd J=6.0 and 0.6 Hz, 1H: CH at position 1'); 5.30 (d, J=6.0 Hz: 1H, OHat position 1'); 8.31 (s, 1H: CH at position 5); 8.53 (s, 1H: CH atposition 3),

HPLC on Y.M.C. 180DS-AQ column of 150×4.6 mm (batch AIT/DE940377)marketed by AIT, isocratic elution H₂ O +0.1% formic acid with a flowrate of 1 ml/min, W detection at 270 nm, retention time: 3 min 61 S.

The hypoglycaemic activity of the mixtures I and II, of sodium oxamateand of the compounds of formula (I) have been determined in mice madediabetic with streptozocin and in normal mice with post-prandialhyperglycaemia according to the following procedures:

I--Mice made diabetic with streptozotocin

Swiss albino mice weighing between 22 and 25 g are made diabetic withstreptozotocin administered by intraperitoneal injection at a dose of210 or 265 mg/Kg of mouse, diluted in a citrate buffer at aconcentration such that each mouse receives 0.2 ml of the solution onday 1 (D1). 3 to 4 days later, a check is made on 2 to 3 mice to see ifthe diabetes is established (glycaemia greater than 7.2 mmol/liter (130mg per 100 ml). The glycaemia is then measured after starving for 4hours. If the mice have become diabetic, they are divided into batchesof 5 to 7 mice. Each of the batches receives, from D1 and daily, aselected dose of product. The administration is made once a day and at afixed time, by gastric intubation and in a volume of 0.4 ml of distilledwater as vehicle. Two batches of controls are made up:

one batch of untreated diabetic mice

one batch or normal mice

These two batches of controls receive 0.4 ml of vehicle by gastricintubation and simultaneously with the treated mice.

The treatment lasts for 4 days. On the 5th day (D5), there is noadministration of product. After fasting for 4 hours, the finalglycaemias are measured.

II--Normal mice with post-prandial hyperglycaemia

Swiss albino mice weighing between 22 and 25 g are made to havepost-prandial hyperglycaemia by the following procedure:

fasting 2 hours

food in excess for 1 hour

fasting 2 hours

The glycaemia is measured at the end of the last two hours of fasting,which constitutes the glycaemia at time T0. The mice are then dividedinto homogeneous batches according to the measured glycaemias. Theproducts to be evaluated are administered without delay by gastricintubation in 0.4 ml of distilled water. The control batch receives theexcipient (0.4 ml of distilled water). After one hour, the finalglycaemias are measured which constitute the glycaemia at time T60.

    __________________________________________________________________________    Results obtained of the glycaemia of mice made diabetic with streptozotoci    PRODUCTS     NUMBER                                                                             GLYCAEMIA AT D1                                                                         GLYCAEMIA AT D5                                     mg/mouse/day OF MICE mg/100 ml mg/100 ml % inhibition                       __________________________________________________________________________    Sodium oxamate (0.5 mg)                                                                    5    153.40 ± 11.53                                                                        89.90 ± 13.94                                                                       -41.46                                    Mixture II (0.5 mg) 5 270.50 ± 58.72 117.50 ± 17.35 -56.56                                                   2-(tetrahydroxybutyl)-5- 5 304.25                                            ± 99.35 164.50 ± 95.13 -45.93       (2',3',4'-trihydroxybutyl)-                                                   pyrazine (0.5 mg)                                                             2-(tetrahydroxybutyl)-5- 6 320.83 ± 130.30 174.00 ± 97.74 -45.77                                             (2',3',4'-trihydroxybutyl)-                                                   pyrazine and 2-(tetrahydroxy-                                                 butyl)-6-(2',3',4'-                      trihydroxybutyl)pyrazine                                                      (50/50) (0.25 mg)                                                             Controls 5 221.66 ± 50.17 210.33 ± 20.07  -5.11                       __________________________________________________________________________

    __________________________________________________________________________    Results obtained on the glycaemia of normal mice with post-prandial           hyperglycaemia                                                                                   NUMBER                                                       PRODUCTS OF GLYCAEMIA AT T0 GLYCAEMIA AT T60                                  mg/mouse/day MICE mg/100 ml mg/100 ml % inhibition                          __________________________________________________________________________    Mixture I (5 mg)    5   129.50 ± 29.06                                                                        87.60 ± 14.16                                                                        -32.35                             Sodium oxamate (0.5 mg) 15 136.46 ± 23.69 102.93 ± 20.95 -24.57                                                     Mixture II (0.5 mg) 15                                                       133.93 ± 17.50 105.60                                                      ± 16.91 -21.15                  2,5-di(tetrahydroxybutyl)pyrazine (0.5 mg) 10 136.25 ± 16.92 102.41                                                   ± 14.12 -24.83                  2-(tetrahydroxybutyl)-5-(2',3',4'-trihydroxy- 10 128.75 ± 21.23                                                       90.50 ± 19.55 -29.70                                                        butyl)pyrazine (0.5 mg)                                                       2-(tetrahydroxybutyl)-5-(2',                                                 3',4'-trihydroxy-  5 126.60                                                   ± 17.03 100.60 ± 16.88                                                  -20.53                             butyl)pyrazine and 2-(tetrahydroxybutyl)-6-                                   (2',3',4'-trihydroxybutyl)pyrazine (50/50)                                    (0.25 mg)                                                                     Controls 10 137.83 ± 13.99 130.50 ± 20.23  -5.32                      __________________________________________________________________________

The mixtures according to the invention, the sodium oxamate and thecompounds of formula (I) have a low toxicity. Their LD₅₀ is greater than2000 mg/kg orally in mice.

The mixtures according to the invention, the sodium oxamate and thecompounds of formula (I) reduce the glycaemia in a diabetic subject andare therefore useful in the treatment of diabetes and the complicationsof diabetes.

When these products are used in monotherapy in the treatment ofdiabetes, there is no risk of hypoglycaemia. They are true antidiabeticagents. It appears that this effect results from a peripheral increasein glucose. The products do not significantly stimulate the secretion ofinsulin but the presence of small quantities of insulin is necessary fortheir action.

In sand rats (Psammomys obesus) which are spontaneously diabetic incaptivity, the products decrease hyperglycaemia, prevent or decreasecataract and restore a degree of fertility.

In human therapy, these products are therefore useful in the preventionand treatment of diabetes and especially type II diabetes (NID diabetes)not exhibiting acetonuria, obese diabetes, diabetes at the age of aboutfifty, metaplethoric diabetes, diabetes affecting the elderly and milddiabetes. They can be used as a supplement to insulin therapy (becauseof their insulin-potentiating activity) in insulin-dependent diabeteswhere they make it possible to gradually reduce the dose of insulin,unstable diabetes, insulin-resistant diabetes, and as a supplement tohypoglycaemic sulphamides when these do not provide a sufficientdecrease in glycaemia. These products can also be used in thecomplications of diabetes such as hyperlipaemias, lipid metabolismdisorders, dyslipaemias and obesity. They are also useful in theprevention and treatment of the lesions of atherosclerosis and theircomplications (coronopathies, myocardial infarction, cardiomyopathies,progression of these three complications into left ventricularinsufficiency, various arteriopathies, arteritis of the lower limbs withclaudication and progression into ulcers and gangrene, cerebral vascularinsufficiency and its complications and sexual impotence of vascularorigin), diabetic retinopathy and all its manifestations (increase incapillary permeability, dilation and capillary thrombosis,microaneurysms, arteriovenous shunt, venous dilation, punctiform andmacular haemorrhages, exudates, macular oedemas, manifestations ofproliferative retinopathy: neovessels, proliferative retinitis scars,haemorrhages of the vitreous body, retinal detachment), diabeticcataract, diabetic neuropathy in its various forms (peripheralpolyneuropathies and its manifestations such as paresthesias,hyperesthesias and pain, mononeuropathies, radiculopathies, autonomousneuropathies, diabetic amyotrophies), the manifestations of diabeticfoot (ulcers of the lower extremities and of the foot), diabeticnephropathy in its two diffuse and nodular forms, atheromatoses (rise inHDL lipoproteins promoting the elimination of cholesterol from theatheroma plaques, decrease in the LDL lipoproteins, decrease in theLDL/HDL ratio, inhibition of oxidation of the LDLs, decrease in plateletadhesiveness), hyperlipaemias and dyslipaemias (hypercholesterolaemias,hypertriglyceridaemias, normalization of the fatty acid level,normalization of the uricaemia, normalization of the A and Bapoproteins), cataracts, high blood pressure and its consequences.

The medicaments according to the invention consist of a mixtureaccording to the invention, sodium oxamate, a compound of formula (I) ora combination of these products, in the pure state or in the form of acomposition in which it is combined with any other pharmaceuticallycompatible product which may be inert or physiologically active. Themedicaments according to the invention can be used orally, parenterally,rectally or topically.

As solid compositions for oral administration, there may be usedtablets, pills, powders (gelatine capsules, cachets) or granules. Inthese compositions, the active ingredient according to the invention ismixed with one or more inert diluents, such as starch, cellulose,sucrose, lactose or silica, under an argon stream. These compositionsmay also comprise substances other than the diluents, for example one ormore lubricants such as magnesium stearate or talc, a colorant, acoating (sugar-coated tablets) or a glaze.

As liquid compositions for oral administration, there may be usedpharmaceutically acceptable solutions, suspensions, emulsions, syrupsand elixirs containing inert diluents such as water, ethanol, glycerol,vegetable oils or paraffin oil. These compositions may comprisesubstances other than the diluents, for example wetting, sweetening,thickening, flavouring or stabilizing products.

The sterile compositions for parenteral administration may preferably besolutions in aqueous or nonaqueous form, suspensions or emulsions. Assolvent or vehicle, there may be used water, propylene glycol,polyethylene glycol, vegetable oils, in particular olive oil, injectableorganic esters, for example ethyl oleate or other suitable organicsolvents. These compositions may also contain adjuvants, in particularwetting, isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be performed in several ways, for example byaseptizing filtration, by incorporating sterilizing agents into thecomposition, by irradiation or by heating. They can also be prepared inthe form of sterile solid compositions which can be dissolved at thetime of use in sterile water or any other injectable sterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active product, excipientssuch as cocoa butter, semisynthetic glycerides or polyethylene glycols.

The compositions for topical administration may be for example creams,lotions, collyria, collutoria, nasal drops or aerosols.

The doses depend on the desired effect, the duration of treatment andthe route of administration used; they are generally between 150 mg and600 mg per day via the oral route for an adult with unit doses rangingfrom 50 mg to 200 mg of active substance.

In general, the doctor will determine the appropriate dosage accordingto the age, weight and all the other factors specific to the subject tobe treated.

The following examples illustrate compositions according to theinvention:

EXAMPLE A

Gelatine capsules in doses of 50 mg of active product having thefollowing composition are prepared according to the usual technique:

    ______________________________________                                        Active product      50        mg                                                Cellulose 18 mg                                                               Lactose 55 mg                                                                 Colloidal silica 1 mg                                                         Sodium carboxymethyl starch 10 mg                                             Talc 10 mg                                                                    Magnesium stearate 1 mg                                                     ______________________________________                                    

EXAMPLE B

Tablets in doses of 50 mg of active product having the followingcomposition are prepared according to the usual technique:

    ______________________________________                                        Active product         50        mg                                             Lactose 104 mg                                                                Cellulose 40 mg                                                               Polyvidone 10 mg                                                              Sodium carboxymethyl starch 22 mg                                             Talc 10 mg                                                                    Magnesium stearate 2 mg                                                       Colloidal silica 2 mg                                                         Hydroxymethylcellulose, glycerine, titanium 245 mg                            oxide (72-3.5-24.5) qs 1 finished film-coated                                 tablet containing                                                           ______________________________________                                    

EXAMPLE C

An injectable solution containing 50 mg of active product having thefollowing composition is prepared:

    ______________________________________                                        Active product   50          mg                                                 Benzoic acid 80 mg                                                            Benzyl alcohol 0.06 ml                                                        Sodium benzoate 80 mg                                                         Ethanol at 95% 0.4 ml                                                         Sodium hydroxide 24 mg                                                        Propylene glycol 1.6 ml                                                       Water qs 4 ml                                                               ______________________________________                                    

We claim:
 1. A mixture I, said mixture being free of polyphenol andsterol derivatives, wherein said mixture is obtained by the processcomprising:grinding Eugenia Jambolana Lamarck seeds into a powder,macerating the powder with a lower aliphatic alcohol while applying heatthereto, filtering the product of said macerating step and recovering aninsoluble part no longer containing polyphenol or sterol compounds,treating the insoluble part with an ammoniacal solution to obtain anammoniacal mass, treating the ammoniacal mass with a lower aliphaticalcohol while applying heat thereto, and filtering the product of saidalcohol treated ammoniacal mass and recovering and drying an insolublematter from said filtered product to obtain a dried mixture I.
 2. Aprocess for the preparation of the mixture I as claimed in claim 1, saidprocess comprising the steps of:a--drying and subsequently grindingEugenia Jambolana Lamarck seeds, screening the ground seeds to obtain apowder, and macerating the powder, with stirring, at a temperatureranging from 40 to 70° C., in a lower aliphatic alcohol having 1 to 4carbon atoms, b--filtering the product obtained from the macerating stepunder vacuum to recover an insoluble substance, c--macerating theinsoluble substance obtained from said filtering step, with stirring, ata temperature ranging from 40 to 70° C., in a lower aliphatic alcoholhaving 1 to 4 carbon atoms, d--filtering the alcoholic solution ofmacerated insoluble substance obtained from step c under vacuum andseparating the alcoholic phase from the insoluble substance, e--takingup the insoluble substance separated in step d in an ammoniacal solutionat a temperature ranging from 10 to 30° C. to obtain a wet ammoniacalmass, f--taking up the wet ammoniacal mass obtained in step e in awater-lower aliphatic alcohol solution having 1 to 4 carbon atoms, at atemperature ranging from 40 to 70° C., g--filtering the alcoholicsolution of wet ammoniacal mass obtained in step f and separating thealcoholic phase therefrom to isolate an insoluble substance from thealcoholic solution, h--washing the insoluble substance isolated in stepg with a lower aliphatic alcohol having 1 to 4 carbons atoms, filteringthe solution of alcohol and insoluble substance and separating thealcoholic phase therefrom to isolate said insoluble substance, andi--recovering and drying the insoluble substance from said filtered andseparated solution of step h to obtain a dried mixture I.
 3. The processof claim 2, wherein the macerating of step a is carried out using 2 to10 liters of a lower aliphatic alcohol per 1 kg of screened powder. 4.The process of claim 2, wherein the macerating of step a is carried outover 1 hour at 60° C. using 5 liters of ethanol with a titre of 93-95°Gay Lussac.
 5. The process of claim 2, wherein the filtration of steps band d is carried out under a vacuum of 40 kPa.
 6. The process of claim2, wherein step c is carried out using 2 to 10 liters of a loweraliphatic alcohol per 1 kg of starting screened powder.
 7. The processof claim 2, wherein step c is carried out over 1 hour at 60° C. using 4liters of ethanol with a titre of 93-95° Gay Lussac.
 8. The process ofclaim 2, wherein step e is carried out using 750 to 1250 ml of anaqueous ammoniacal solution per 1 kg of starting screened powder.
 9. Theprocess of claim 2, wherein step e is carried out at a temperature ofabout 20° C. for 10 to 30 hours using 1 liter, per 1 kg of startingscreened powder, of aqueous ammoniacal solution containing 350 ml of 28%ammonium hydroxide.
 10. The process of claim 2, wherein the wetammoniacal mass of step f is taken up in 2 to 10 liters of a water-loweraliphatic alcohol solution per 1 kg of starting screened powder.
 11. Theprocess of claim 2, wherein step f is carried out in 5 liters of anethanol-water mixture at 60° C. for 1 hour, wherein the mixture contains75% of ethanol and 25% of water by volume.
 12. The process of claim 2,wherein the washing of step h is carried out using 500 to 1500 ml of alower aliphatic alcohol per 1 kg of starting screened powder.
 13. Theprocess of claim 2, wherein the washing of step h is carried out using 1liter of ethanol, and the filtering of step h is carried out on a cottoncloth and under a vacuum of about 80 kPa.
 14. The process of claim 2,wherein the drying of step i is carried out in the open air andprotected from light.
 15. A mixture II, said mixture II being free ofpolyphenol and sterol derivatives, wherein said mixture II is obtainedby the process comprising:treating the mixture I of claim 1 with awater-lower aliphatic alcohol solution, filtering the product obtainedfrom the treating step and partially concentrating the filtrate,purifying the partially concentrated filtrate on at least one non-polaradsorbent resin, partially concentrating the product obtained from thepurification step, and subjecting the partially concentrated material tocentrifugation and ultrafiltration to isolate the mixture II.
 16. Aprocess for the preparation of the mixture II as claimed in claim 15,said process comprising the steps of:a--drying and subsequently grindingEugenia Jambolana Lamarck seeds, screening the ground seeds to obtain apowder, and macerating the powder, with stirring, at a temperatureranging from 40 to 70° C., in a lower aliphatic alcohol having 1 to 4carbon atoms, b--filtering the product obtained from the macerating stepunder vacuum to recover an insoluble substance, c--macerating theinsoluble substance obtained from said filtering step, with stirring, ata temperature ranging from 40 to 70° C., in a lower aliphatic alcoholhaving 1 to 4 carbon atoms, d--filtering the alcoholic solution ofmacerated insoluble substance obtained from step c under vacuum andseparating the alcoholic phase from the insoluble substance, e--takingup the insoluble substance separated in step d in an ammoniacal solutionat a temperature ranging from 10 to 30° C. to obtain a wet ammoniacalmass, f--taking up the wet ammoniacal mass obtained in step e in awater-lower aliphatic alcohol solution having 1 to 4 carbon atoms, at atemperature ranging from 40 to 70° C., g--filtering the alcoholicsolution of wet ammoniacal mass obtained in step f and separating thealcoholic phase therefrom to isolate an insoluble substance from thealcoholic solution, h--washing the insoluble substance isolated in stepg with a lower aliphatic alcohol having 1 to 4 carbons atoms, filteringthe solution of alcohol and insoluble substance and separating thealcoholic phase therefrom to isolate said insoluble substance, andi--recovering and drying the insoluble substance from said filtered andseparated solution of step h to obtain a dried mixture I, j--treatingthe mixture I obtained from step i with a water-lower aliphatic alcoholsolution having 1 to 4 carbon atoms, k--decanting the product of step jto obtain a top phase and a bottom phase, drawing off and filtering thetop phase to give a filtrate 1; treating the bottom phase with water andfiltering the resulting aqueous solution to give a filtrate 2; poolingthe filtrates 1 and 2, and concentrating the pooled filtrates to anaqueous phase, l--treating the aqueous phase obtained from step k withat least one nonpolar adsorbent resin and filtering the resultingmaterial, m--concentrating the filtrate obtained from step I, thensubjecting the resulting concentrated substance to filtration and thenultrafiltration, and n--freeze-drying and isolating a material that ismixture II.
 17. The process of claim 16, wherein step j is carried outusing 10 to 25 liters of a water-lower aliphatic alcohol solution per 1kg of mixture I, wherein the alcohol solution contains from 95% to 90%of water and from 5% to 10% of lower aliphatic alcohol by volume. 18.The process of claim 16, wherein step j is carried out using 18 litersof a water-ethanol solution, wherein the solution comprises 17.7 litersof water and 0.93 liter of ethanol.
 19. The process of claim 16, whereinin step k the top phase is filtered on a cotton cloth, 10 to 25 litersof water are added to the bottom phase per 1 kg of mixture I, and themixture of the water and the bottom phase is filtered on sintered glass.20. The process of claim 16, wherein the concentrating of step k iscarried out in a thermosiphon concentrator at a temperature of 35° C.under a vacuum of 0.4 kPa.
 21. The process of claim 16, wherein in stepI, a S861 resin or XAD-type resin marketed by Rhom and Hass is used andthe filtering is performed using sintered glass.
 22. The process ofclaim 16, wherein in step m, the concentrating is carried out in athermosiphon concentrator at a temperature of 35° C. under a vacuum of0.4 kPa.
 23. The process of claim 16, wherein in step m, threesuccessive ultrafiltrations are carried out on 10 kDa, 3 kDa and 1 kDacartridges.
 24. A process for preparing sodium oxamate or a compound offormula: ##STR3## in which R₁ represents a hydrogen atom and R₂represents a chain of formula: ##STR4## or R₁ represents a chain offormula (A) and R₂ represents a hydrogen atom, said process comprisingthe steps of:o--chromatographing the mixture II of claim 15 on aninfusorial earth column to produce fractions containing four products,recovering the fractions containing four products, and pooling thesefractions into a single fraction, and p--chromatographing the pooledsingle fraction obtained from step o on a Sephadex® column in order toobtain sodium oxamate, the compound of formula (I) for which R₁represents a hydrogen atom and R₂ represents a residue (B), and amixture of the compound of formula (I) for which R₁ represents ahydrogen atom and R₂ represents a residue (A) and of the compound offormula (I) for which R₁ represents a residue (A) and R₂ represents ahydrogen atom.
 25. The process of claim 24, wherein the chromatographingof step o is carried out on a CHEM ELUT® column marketed by Prolabo,wherein said column is saturated with water and then eluted successivelywith (1) heptane, (2) a heptane-ethyl acetate mixture containing 50% ofheptane and 50% of ethyl acetate by volume, (3) an ethylacetate-n-butanol mixture progressing from 95% to 90%, 80%, 50%, 20% and0% of ethyl acetate by volume, and (4) an n-butanol-water mixture having98% then 95% of n-butanol by volume.
 26. The process of claim 24,wherein the chromatographing of step p is carried out using awater-ethanol mixture having 50% of water and 50% of ethanol by volume.27. The process of claim 24, further comprising thestep:q--chromatographing of the mixture of the compound of formula (I)for which R₁ represents a hydrogen atom and R₂ represents a residue (A)and of the compound of formula (I) for which R₁ represents a residue (A)and R₂ represents a hydrogen atom by HPLC.
 28. The process of claim 27,wherein the chromatographing of step q is carried out on a YMC 180DS-AQcolumn marketed by AIT with, as eluent, a water containing 0.1% offormic acid mixture.
 29. A pharmaceutical composition comprising themixture I as claimed in claim 1, together with a pharmaceuticallyacceptable carrier.
 30. A pharmaceutical composition comprising themixture II as claimed in claim 15, together with a pharmaceuticallyacceptable carrier.
 31. A method for the treatment or prevention ofdiabetes or a complication of diabetes, comprising administering to ahost in need of such treatment or prevention an effective amount of themixture I as claimed in claim
 1. 32. A method for the treatment orprevention of diabetes or a complication of diabetes, comprisingadministering to a host in need of such treatment or prevention aneffective amount of the mixture II as claimed in claim
 15. 33. A methodfor the treatment or prevention of diabetes or a complication ofdiabetes, comprising administering to a host in need of such treatmentor prevention the pharmaceutical composition as claimed in claim
 29. 34.A method for the treatment or prevention of diabetes or a complicationof diabetes, comprising administering to a host in need of suchtreatment or prevention the pharmaceutical composition as claimed inclaim 30.